The role of microglia and macrophages in glioma maintenance and progression

There is a growing recognition that gliomas are complex tumors composed of neoplastic and non-neoplastic cells, which each individually contribute to cancer formation, progression and response to treatment. The majority of the non-neoplastic cells are tumor-associated macrophages (TAMs), either of peripheral origin or representing brain-intrinsic microglia, that create a supportive stroma for neoplastic cell expansion and invasion. TAMs are recruited to the glioma environment, have immune functions, and can release a wide array of growth factors and cytokines in response to those factors produced by cancer cells. In this manner, TAMs facilitate tumor proliferation, survival and migration. Through such iterative interactions, a unique tumor ecosystem is established, which offers new opportunities for therapeutic targeting

Developmental changes in the membrane current pattern, K+ buffer capacity, and morphology of glial cells in the corpus callosum slice

Recent studies indicated that glial cells in tissue culture can express a variety of different voltage-gated channels, while little is known about the presence of such channels in glial cells in vivo. We used a mouse corpus callosum slice preparation, in which after postnatal day 5 (P5) more than 99% of all perikarya belong to glial cells (Sturrock, 1976), to study the current patterns of glial cells during their development in situ. We combined the patch-clamp technique with intracellular labeling using Lucifer yellow (LY) and subsequent ultrastructural characterization. In slices of mice from P6 to P8, we predominantly found cells expressing delayed-rectifier K+ currents. They were similar to those described for cultured glial precursor cells (Sontheimer et al., 1989). A-type K+ currents or Na+ currents were not or only rarely observed, in contrast to cultured glial precursors. LY labeling revealed that numerous thin processes extended radially from the perikaryon of these cells, and ultrastructural observations suggested that they resemble immature glial cells. In slices of older mice (P10-13), when myelination of the corpus callosum has already commenced, many cells were characterized by an almost linear current-voltage relationship. This current pattern was similar to cultured oligodendrocytes (Sontheimer et al., 1989). Most processes of LY-filled cells with such a current profile extended parallel to each other. Electron microscopy showed that these processes surround thick, unmyelinated axons. We suggest that cells with oligodendrocyte-type electrophysiology are promyelinating oligodendrocytes. In contrast to cultured oligodendrocytes, membrane currents of promyelinating oligodendrocytes in the slice decayed during the voltage command. This decay was due not to inactivation, but to a marked change in the potassium equilibrium potential within the voltage jump. This implies that, in the more mature corpus callosum, small membrane polarizations in a physiological range can lead to extensive changes in the K+ gradient across the glial membrane within a few milliseconds

Membrane properties of ameboid microglial cells in the corpus callosum slice from early postnatal mice

Microglial cells in culture are distinct from neurons, macroglial cells, and macrophages of tissues other than brain with respect to their membrane current pattern. To assess these cells in the intact tissue, we have applied the patch-clamp technique to study membrane currents in microglial cells from acute, whole brain slices of 6-9-d-old mice in an area of microglial cell invasion, the cingulum. As strategies to identify microglial cells prior to or after recording, we used binding and incorporation of Dil-acetylated low-density lipoproteins, binding of fluorescein isothiocyanate-coupled IgG via microglial Fc-receptors, and ultrastructural characterization. As observed previously for cultured microglial cells, depolarizing voltage steps activate only minute if any membrane currents, while hyperpolarizing voltage steps induced large inward currents. These currents exhibited properties of the inwardly rectifying K+ channel in that the reversal potential depended on the transmembrane K+ gradient, inactivation time constants decreased with hyperpolarization, and the current was blocked by tetraethylammonium (50 mM). This study represents the first attempt to assess microglial cells in situ using electrophysiological methods. It opens the possibility to address questions related to the function of microglial cells in the intact CNS

Different mechanisms promote astrocyte Ca2+ waves and spreading depression in the mouse neocortex

Cortical spreading depression (CSD) is thought to play an important role in different pathological conditions of the human brain. Here we investigated the interaction between CSD and Ca2+ waves within the astrocyte population in slices from mouse neocortex (postnatal days 10-14). After local KCl ejection as a trigger for CSD, we recorded the propagation of Ca2+ increases within a large population of identified astrocytes in synchrony with CSD measured as intrinsic optical signal (IOS) or negative DC-potential shift. The two events spread with 39.2 +/- 3.3 mum/sec until the IOS and negative DC-potential shift decayed after approximately 1 mm. However, the astrocyte Ca2+ wave continued to propagate for up to another 500 microm but with a reduced speed of 18.3 +/- 2.5 microm/sec that is also typical for glial Ca2+ waves in white matter or culture. While blocking CSD using MK-801 (40 microm), an NMDA-receptor antagonist, the astrocyte Ca2+ wave persisted with a reduced speed (13.2 +/- 1.5 microm/sec). The specific gap junction blocker carbenoxolon (100 microm) did not prevent CSD but decelerated the speed (2.9 +/- 0.9 microm/sec) of the astrocyte Ca2+ wave in the periphery of CSD. We also found that interfering with intracellular astrocytic Ca2+ signaling by depletion of internal Ca2+ stores does not affect the spread of the IOS. We conclude that CSD determines the velocity of an accompanying astrocytic Ca2+ response, but the astrocyte Ca2+ wave penetrates a larger territory and by this represents a self-reliant phenomenon with a different mechanism of propagation

Experimental cortical spreading depression induces NMDA receptor dependent potassium currents in microglia

Cortical spreading depression (CSD) is a propagating event of neuronal depolarization, which is considered as the cellular correlate of the migraine aura. It is characterized by a change in the intrinsic optical signal and by a negative DC potential shift. Microglia are the resident macrophages of the CNS and act as sensors for pathological changes. In the present study, we analyzed whether microglial cells might sense CSD by recording membrane currents from microglia in acutely isolated cortical mouse brain slices during an experimentally induced CSD. Coincident with the change in the intrinsic optical signal and the negative DC potential shift we recorded an increase in potassium conductance predominantly mediated by K(+) inward rectifier (Kir)2.1, which was blocked by the NMDA receptor antagonist D-AP5. Application of NMDA and an increase in extracellular K(+) mimics the CSD-induced Kir activation. Application of D-AP5, but not the purinergic receptor antagonist RB2, blocks the NMDA-induced Kir activation. The K(+) channel blocker Ba(2+) blocks both the CSD- and the NMDA-triggered increase in Kir channel activity. In addition, we could confirm previous findings that microglia in the adult brain do not express functional NMDA receptors by recording from microglia cultured from adult brain. From these observations we conclude that CSD activates neuronal NMDA receptors, which lead to an increase in extracellular [K(+)] resulting in the activation of Kir channel activity in microglia

Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release function

Microglia-brain macrophages are immune-competent cells of the CNS and respond to pathologic events. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration ([Ca 2+]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of basal [Ca 2+]i along with a suppression of evoked calcium signaling, as indicated by reduced [Ca 2+]i transients during stimulation with UTP and complement factor 5a. Presence of the calcium chelator BAPTA prevented the activation-associated changes in [Ca 2+]i and restored much of the signaling efficacy. We also evaluated downstream consequences of a basal [Ca 2+]i lifting during microglial activation and found BAPTA to strongly attenuate the LPS-induced release of nitric oxide (NO) and certain cytokines and chemokines. Furthermore, microglial treatment with ionomycin, an ionophore elevating basal [Ca 2+]i, mimicked the activation-induced calcium signal suppression but failed to induce release activity on its own. Our findings suggest that chronic elevation of basal [Ca 2+]i attenuates receptor-triggered calcium signaling. Moreover, increased [Ca 2+]i is required, but by itself is not sufficient, for release of NO and certain cytokines and chemokines. Elevation of basal [Ca 2+]i could thus prove a central element in the regulation of executive functions in activated microglia

Developmental regulation of voltage-gated K+ channel and GABAA receptor expression in Bergmann glial cells

Bergmann glial cells are closely associated with neurons: during development they provide guiding structures for migrating granule cells and in the adult cerebellum they display intimate interactions with Purkinje cells. In this study, we have addressed the question of whether such changes in neuronal-glial interactions during development are accompanied by variations in the membrane properties of Bergmann glial cells. We used a mouse cerebellum slice preparation to study membrane currents of the Bergmann glial cells at various stages of development in situ using the patch-clamp technique. The distinct morphology of Bergmann glial cells was revealed by Lucifer yellow injections during recording. While Bergmann glial cells in mice of postnatal day 20 (P20) to P30 have thick processes with arborized, irregularly shaped leaf-like appendages, the processes of cells from younger mice (P5-P7) are thinner and smoother. This morphological maturation is accompanied by a variation in voltage-gated currents. In cells from P5 to P7, delayed outward- and inward-rectifying K+ currents were recorded, while older Bergmann glial cells were characterized by, large, voltage- and time-independent K+ currents. In addition, application of GABA induces two effects, a rapid activation of a Cl- conductance and a longer-lasting decrease in the (resting) K+ conductance. Both effects were mediated by benzodiazepine-insensitive GABAA receptors. Responses in cells of P5-P7 mice were large as compared to the small or even undetectable responses in P20-P30 cells. These GABAA receptors were characterized immunohistochemically in mice and rat brain sections with five subunit-specific antibodies. Bergmann glial cells exhibit a distinct but transient immunoreactivity for the GABAA receptor alpha 2-, alpha 3-, and delta-subunits. Staining is maximal between P7 and P10 and decreases gradually thereafter. In contrast, antibodies to the alpha 1- and beta 2,3-subunits fail to decorate Bergmann glial cells, although they yield a prominent staining of both the Purkinje cells and the granule cells. These changes in the Bergmann glial cell membrane properties and GABAA receptor expression suggest a transition between functional states during development of the Bergmann glial cells

TLR2 controls random motility, while TLR7 regulates chemotaxis of microglial cells via distinct pathways

Microglial cells are the pathologic sensor of the brain, and any pathologic event triggers microglial activation, which involves migration of these cells to a lesion site. Employing different migration assays, we show that ligands for toll-like receptor (TLR) 2 stimulate random motility, while TLR7 ligands are chemoattractants. The subtype specificity of the TLR ligands was verified by using different TLR-deficient (TLRKO) mouse lines. PI3K and Rac inhibition impairs both TLR2- and TLR7-stimulated microglial migration. In contrast, Akt phosphorylation is only required for the TLR2-, but not for the TLR7-stimulated pathway. Interestingly, P2Y12 receptor signaling is involved in the TLR2 activation-induced microglial migration but not TLR7. Furthermore, TLR7 mRNA expression is down-regulated by TLR2 and TLR7 activation. We conclude that TLRs control the migratory behavior of microglia in a distinct manner

Mechanisms of C5a and C3a complement fragment-induced [Ca2+]i signaling in mouse microglia

Microglial cells are activated in response to brain insults; the mechanisms of this process are not yet understood. One of the important signaling mechanisms that might be involved in microglia activation is related to changes in the intracellular calcium concentration ([Ca2+]i). Using fluo-3 microfluorimetry, we have found that external application of the complement fragment C5a (4-10 nM) induced [Ca2+]i elevation in microglial cells in situ in corpus callosum slices. Similarly, application of complement fragments C5a (0.1-10.0 nM) or C3a (100 nM) generates biphasic [Ca2+]i transients composed of an initial peak followed by a plateau in cultured microglia. Incubation of microglial cells for 30 min with pertussis toxin (PTX; 1 microgram/ml) inhibited both C5a- and C3a-triggered [Ca2+]i responses, suggesting the involvement of PTX-sensitive G-proteins in the signal transduction chain. Removal of Ca2+ ions from the extracellular solution eliminated the plateau phase and limited the response to the initial peak. The restoration of the extracellular Ca2+ concentration within 30-60 sec after the beginning of the complement fragment-induced [Ca2+]i elevation led to the recovery of the plateau phase. Inhibition of the endoplasmic reticulum Ca2+ pumps with 500 nM thapsigargin transiently increased the [Ca2+]i and blocked the [Ca2+]i signals in response to subsequent complement fragment application. Our data suggest that complement factors induce [Ca2+]i responses by Ca2+ release from internal pools and subsequent activation of Ca2+ entry controlled by the filling state of the intracellular Ca2+ depots

The subpopulation of microglia sensitive to neurotransmitters/neurohormones is modulated by stimulation with LPS, interferon-γ, and IL-4

Recently, neurotransmitters/neurohormones have been identified as factors controlling the function of microglia, the immune competent cells of the central nervous system. In this study, we compared the responsiveness of microglia to neurotransmitters/neurohormones. We freshly isolated microglia from healthy adult C57Bl/6 mice and found that only a small fraction (1-20%) responded to the application of endothelin, histamine, substance P, serotonin, galanin, somatostatin, angiotensin II, vasopressin, neurotensin, dopamine, or nicotine. In cultured microglia from neonatal and adult mice, a similarly small population of cells responded to these neurotransmitters/neurohormones. To induce a proinflammatory phenotype, we applied lipopolysaccaride (LPS) or interferon-gamma (IFN-{gamma}) to the cultures for 24 h. Several of the responding populations increased; however, there was no uniform pattern when comparing adult with neonatal microglia or LPS with IFN-{gamma} treatment. IL-4 as an anti-inflammatory substance increased the histamine-, substance P-, and somatostatin-sensitive populations only in microglia from adult, but not in neonatal cells. We also found that the expression of different receptors was not strongly correlated, indicating that there are many different populations of microglia with a distinct set of receptors. Our results demonstrate that microglial cells are a heterogeneous population with respect to their sensitivity to neurotransmitters/neurohormones and that they are more responsive in defined activation states